The invention relates generally to the field of vaccine development. More particularly, the invention relates to the development of prophylactic and therapeutic vaccines effective against intracellular pathogens.
The development of vaccines directed against intracellular pathogens, for example, viruses, bacteria, protozoa, fungi, and intracellular parasites, is ongoing. The development and use of vaccines has proved invaluable in preventing the spread of disease in man. For example, in 1967, smallpox was endemic in 33 countries with 10 to 15 million cases being reported annually. At that time, the World Health Organization introduced a program to eradicate smallpox. Approximately one decade later, smallpox was successfully eradicated from the human population.
Theoretically, an ideal vaccine has a long shelf life, is capable of inducing with a single dose long lasting immunity against a preselected pathogen and all of its phenotypic variants, is incapable of causing the disease to which the vaccine is directed against, is effective therapeutically and prophylactically, is prepared easily and economically using standard methodologies, and can be administered easily in the field.
Presently four major classes of vaccine have been developed against mammalian diseases. These include: live-attenuated vaccines; non living whole vaccines; vector vaccines; and subunit vaccines. Several reviews discuss the preparation and utility of these classes of vaccines. See for example, Subbarao et al. (1992) in Genetically Engineered Vaccines, edited by Ciardi et al., Plenum Press, New York; and Melnick (1985) in High Technology Route to Virus Vaccines, edited by Deesman et al., published by the American Society for Microbiology, the disclosures of which are incorporated herein by reference. A summary of the advantages and disadvantages of each of the four classes of vaccines is set forth below.
Live attenuated vaccines comprise live but attenuated pathogens, i.e., non-virulent pathogens, that have been xe2x80x9ccrippledxe2x80x9d by means of genetic mutations. The mutations prevent the pathogens from causing disease in the recipient or vaccinee. The primary advantage of this type of vaccine is that the attenuated organism stimulates the immune system of the recipient in the same manner as the wild type pathogen by mimicking the natural infection. Furthermore, the attenuated pathogens replicate in the vaccinee thereby presenting a continuous supply of antigenic determinants to the recipient""s immune system. As a result, live vaccines can induce strong, long lasting immune responses against the wild type pathogen. In addition, live vaccines can stimulate the production of antibodies which neutralize the pathogen. Also they can induce resistance to the pathogen at its natural portal of entry into the host. To date, live attenuated vaccines have been developed against: smallpox; yellow fever; measles; mumps; rubella; poliomyelitis; adenovirus; and tuberculosis.
Live attenuated vaccines, however, have several inherent problems. First, there is always a risk that the attenuated pathogen may revert back to a virulent phenotype. In the event of phenotypic reversion, the vaccine may actually induce the disease it was designed to provide immunity against. Second, it is expensive and can be impractical to develop live vaccines directed against pathogens that continuously change their antigenic determinants. For example, researchers have been unable to develop a practical live vaccine against the influenza virus because the virus continually changes the antigenic determinants of its coat proteins. Third, live attenuated vaccines may not be developed against infections caused by retroviruses and transforming viruses. The nucleic acids from these viruses may integrate into the recipients genome with the potential risk of inducing cancer in the recipient. Fourth, during the manufacture of live attenuated vaccines adventitious agents present in the cells in which the vaccine is manufactured may be copurified along with the attenuated pathogen. Alien viruses that have been detected in vaccine preparations to date include the avian leukosis virus, the simian papovavirus SV40, and the simian cytomegalovirus. Fifth, live vaccine preparations can be unstable therefore limiting their storage and use in the field. Presently, attempts are being made to develop stabilizing agents which enhance the longevity of the active vaccines.
Non living whole vaccines comprise non viable whole organisms. The pathogens are routinely inactivated either by chemical treatment, i.e., formalin inactivation, or by treatment with lethal doses of radiation. Non living whole vaccines have been developed against: pertussis; typhus; typhoid fever; paratyphoid fever; and particular strains of influenza.
In principle, non living vaccines usually are safe to administer because it is unlikely that the organisms will cause disease in the host. Furthermore, since the organism is dead the vaccines tend to be stable and have long shelf lives. There are, however, several disadvantages associated with non living whole vaccines. First, considerable care is required in their manufacture to ensure that no live pathogens remain in the vaccine. Second, vaccines of this type generally are ineffective at stimulating cellular responses and tend to be ineffective against intracellular pathogens. Third, the immunity elicited by non viable vaccines is usually short-lived and must be boosted at a later date. This process repeatedly entails reaching the persons in need of vaccination and also raises the concern about hypersensitizing the vaccinee against the wild type pathogen.
Vector vaccines, also known as live recombinant vehicle vaccines, may be prepared by incorporating a gene encoding a specific antigenic determinant of interest into a living but harmless virus or bacterium. The harmless vector organism is in turn to be injected into the intended recipient. In theory, the recombinant vector organism replicates in the host producing and presenting the antigenic determinant to the host""s immune system. It is contemplated that this type of vaccine will be more effective than the non-replicative type of vaccine. For such a vaccine to be successful, the vector must be viable, and be either naturally non-virulent or have an attenuated phenotype.
Currently preferred vectors include specific strains of: vaccinia (cowpox) virus, adenovirus, adeno-associated virus, salmonella and mycobacteria. Live strains of vaccinia virus and mycobacteria have been administered safely to humans in the form of smallpox and tuberculosis (BCG) vaccines, respectively. They have been shown to express foreign proteins and exhibit little or no conversion into virulent phenotypes. Several types of vector vaccines using the BCG vector currently are being developed against the human immunodeficiency virus (HIV). For example, the HIV antigenic proteins: gag; env; HIV protease; reverse transcriptase; gp120 and gp41 have been introduced, one at a time, into the BCG vector and shown to induce T cell mediated immune responses against the HIV proteins in animal models (Aldovini et al. (1991) Nature 351:479-482; Stover et al. (1991) Nature 351:456-460; Colston (1991) Nature 351:442-443).
Vector vaccines are capable of carrying a plurality of foreign genes thereby permitting simultaneous vaccination against a variety of preselected antigenic determinants. For example, researchers have engineered several HIV genes into the vaccinia virus genome thereby creating multivalent vaccines which therefore are, in theory, capable of simultaneously stimulating a response against several HIV proteins.
There are several disadvantages associated with vector vaccines. First, it is necessary to identify suitable strains of viable but non-pathogenic organisms that may act as carriers for the genes of interest. Second, vector vaccines can be prepared only when a potentially protective antigenic determinants has been identified and characterized. Accordingly, vector vaccines cannot be prepared against pathogens whose antigenic determinant has not yet been identified or are so variable that the prospect of identifying the antigenic determinant for each variant is impractical. Third, the genes encoding the preselected antigenic determinant must be stably transfected and expressed in the preferred carrier organism. Consequently, the methodologies required for developing this type of vaccine are both labor intensive and time consuming. Fourth, it has not yet been established that recombinant vector vaccines effectively immunize a recipient against a preselected pathogen.
Subunit vaccines usually comprise a subcellular component purified from the pathogen of interest. Subunit vaccines usually are safe to administer because it is unlikely that the subcellular components will cause disease in the recipient. The purified subcellular component may be either a defined subcellular fraction, purified protein, nucleic acid or polysaccharide having an antigenic determinant-capable of stimulating an immune response against the pathogen. The antigenic components can be purified from a preparation of disrupted pathogen. Alternatively, the antigenic proteins, nucleic acids or polysaccharides may be synthesized using procedures well known in the art. Diseases that have been treated with subunit type vaccines include: cholera; diphtheria; hepatitis type B; poliomyelitis; tetanus; and specific strains of influenza.
There are, however, several disadvantages associated with subunit vaccines. First, it is important to identify and characterize the protective antigenic determinant. This can be a labor intensive and time consuming process. As a result it may be impractical to develop subunit vaccines against pathogens with highly variable antigenic determinants. Second, subunit vaccines generally are ineffective at stimulating cytotoxic T cell responses and so they may be ineffective at stimulating an immune response against intracellular pathogens. Third, the immunity elicited by subunit vaccines is usually short-lived, and like the non living whole vaccines must be boosted at a later date therefore raising the concern about hypersensitizing the vaccinee against the wild type pathogen.
Heretofore, many of the inactivated whole and subunit vaccines have not been sufficiently immunogenic by themselves to induce strong, protective responses. As a result, immunostimulants including, for example, aluminum hydroxide; intact mycobacteria; and/or mycobacterial components have been co-administered with these vaccines to enhance the immune response stimulated by the vaccine. Recently, experiments have shown that mycobacterial heat shock proteins may act as carriers for peptide vaccines thereby enhancing the immunogenidty of the peptides in vivo (Lussow et al. (1991) Eur. J. Immunol. 21:2297-2302). Further studies have shown that administering a composition to mice comprising an antigenic peptide chemically crosslinked to a purified mycobacterial stress protein stimulates a humoral (antibody mediated) rather than a temporal (cell mediated) response against the antigenic peptide (Barrios et al. (1992) Eur. J. Immunol. 22:1365-1372).
However, because it is generally believed that cellular responses are required for immunizing against intracellular pathogens (see for example, xe2x80x9cAdvanced Immunology,xe2x80x9d Male et al. (1991) Gower Medical Publishing; Raychaudhuri et al. (1993) Immunology Today 14: 344-348) it is contemplated that conventional subunit and inactivated whole organism vaccines may be ineffective at stimulating immune responses, specifically cytotoxic T cell responses, against intracellular pathogens.
It is an object of the instant invention to provide a safe subunit vaccine comprising a stress protein-peptide complex for administration to a mammal that is capable of inducing, by means of a cytotoxic T cell response, resistance to infection by a preselected intracellular pathogen. The vaccines prepared in accordance with the invention may be used to elicit an immune response against an intracellular pathogens whose antigenic determinants have been identified, have not yet been identified, or where it is impractical to isolate and characterize each of the antigenic determinants. The vaccines prepared in accordance with the invention may be prophylactically and therapeutically effective against preselected pathogens.
Another object of the invention is to provide a method for inducing in a mammal resistance to infection by an intracellular pathogen by administering to the mammal a stress protein-peptide subunit vaccine. Another object is to provide a method for rapidly and cost effectively producing commercially feasible quantities of the stress protein-peptide vaccines from a cell or cell line infected with the intracellular pathogen or alternatively from a cell or cell line transfected with, and expressing a gene encoding a specific antigenic determinant. Still another object is to provide a method for preparing an immunogenic stress protein-peptide subunit vaccine by reconstituting in vitro immunologically unreactive stress proteins and peptides thereby to produce immunoreactive complexes capable of stimulating an immune response against a preselected intracellular pathogen.
These and other objects and features of the invention will be apparent from the description, drawings, and claims which follow.
It has now been discovered that a subunit vaccine containing a stress protein-peptide complex when isolated from cells infected with a preselected intracellular pathogen and then administered to a mammal can effectively stimulate cellular immune responses against cells infected with the same pathogen. Specifically, the immune response is mediated through the cyto toxic T cell cascade which targets and destroys cells containing intracellular pathogens.
The vaccines prepared in accordance with the methodologies described herein provide an alternative approach for stimulating cellular immunity thereby obviating the use of live (attenuated or otherwise) intracellular pathogens. In addition, the vaccines described herein are ideal for inducing immune responses against intracellular pathogens having either defined or as yet undefined immunogenic determinants. Furthermore the vaccines may be used to induce immune responses against intracellular pathogens whose antigenic determinants are either diverse or constantly changing thereby making the isolation and characterization of antigenic determinants impractical.
In a preferred aspect, the invention comprises a vaccine that can be administered to a mammal for inducing in the mammal a cytotoxic T cell response against a preselected intracellular pathogen. Also, it is contemplated that the vaccines may induce in the mammal, by means of a cytotoxic T cell response, resistance to infection by the preselected intracellular pathogen. The vaccines manufactured in accordance with the principles described herein contain an immunogenic stress protein-peptide complex that is capable of stimulating in the recipient a cytotoxic T cell response directed against cells infected with the pathogen of interest. The complex when combined with a pharmaceutically acceptable carrier, adjuvant, or excipient may be administered to a mammal using techniques well known in the art.
The term xe2x80x9cvaccinexe2x80x9d, as used herein, is understood to mean any composition containing a stress protein-peptide complex having at least one antigenic determinant which when administered to a mammal stimulates in the mammal an immune response against the antigenic determinant.
The term xe2x80x9cstress proteinxe2x80x9d as used herein, is understood to mean any cellular protein which satisfies the following criteria. It is a protein whose intracellular concentration increases when a cell is exposed to stressful stimuli, is capable of binding other proteins or peptides, and is capable of releasing the bound proteins or peptides in the presence of adenosine triphosphate (ATP) or low pH. Stressful stimuli include, but are not limited to, heat shock, nutrient deprivation, metabolic disruption, oxygen radicals, and infection with intracellular pathogens.
It will be apparent to the artisan upon reading this disclosure that other recombinant stress proteins, including non native forms, truncated analogs, muteins, fusion proteins as well as other proteins capable of mimicking the peptide binding and immunogenic properties of a stress protein may be used in the preparation of stress protein-peptide vaccines disclosed herein.
The first stress proteins to be identified were the heat shock proteins (Hsp). As their name suggests, Hsps are induced by a cell in response to heat shock. Three major families of Hsp have been identified and are called Hsp60, Hsp70 and Hsp90 because of their respective molecular weights of about 60,70, and 90 kD. Many members of these families subsequently were found to be induced in response to other stressful stimuli, such as those mentioned above.
Stress proteins are found in all prokaryotes and eukaryotes and exhibit a remarkable level of evolutionary conservation. For example, DnaK, the Hsp70 from E. coli has about 50% amino acid sequence identity with Hsp70 proteins from eukaryotes (Bardwell et al. (1984) Proc. Natl. Acad. Sci. 81:848-852). The Hsp60 and Hsp90 families also exhibit similarly high levels of intrafamilial conservation (Hickey et al. (1989) Mol. Cell Biol. 9:2615-2626; Jindal (1989) Mol. Cell. Biol. 9:2279-2283). In addition, it has been discovered that the Hsp-60, Hsp-70, and Hsp-90 families are composed of proteins that are related to the stress proteins in sequence, for example, having greater than 35% amino acid identity, but whose expression levels typically remain unaltered under conditions stressful to the host cell. An example of such a protein includes the constitutively expressed cytosolic protein Hsc 70 to which is related in amino acid sequence to the stress-induced protein Hsp 70. Accordingly, it is contemplated the definition of stress protein, as used herein, embraces other proteins, muteins, analogs, and variants thereof having at least 35% to 55%, preferably 55% to 75%, and most preferably 75% to 95% amino acid identity with members of the three families whose expression levels in a cell are stimulated in response to stressful stimuli.
The term xe2x80x9cpeptidexe2x80x9d, as used herein, is understood to mean any amino acid sequence that is present in a eukaryotic cell infected with an intracellular pathogen but which is not present in a similar cell when the cell is not infected with the same pathogen. The definition embraces peptides that not only originate from the pathogen itself but also peptides which are synthesized by the infected cell in response to infection by the intracellular pathogen.
The term xe2x80x9cimmunogenic stress protein-peptide complexxe2x80x9d, as used herein, is understood to mean any complex containing a stress protein and a peptide that is capable of inducing an immune response in a mammal. The peptides preferably are non covalently associated with the stress protein. The complexes may include, but are not limited to, Hsp60-peptide, Hsp70-peptide and Hsp90-peptide complexes. In a preferred aspect of the invention a stress protein belonging to the Hsp90 family, namely gp96 can be used to generate an effective vaccine containing a gp96-peptide complex. Since the peptides can be dissociated from the complex in the presence of ATP or low pH potentially antigenic peptides can be isolated from cells infected with a preselected intracellular pathogen. Consequently, the antigenic determinants for potentially any intracellular pathogen of interest can be identified readily using the methodologies described herein.
The term xe2x80x9ccytotoxic T cellxe2x80x9d, as used herein, is understood to mean any T lymphocyte expressing the cell surface glycoprotein marker CD8 that is capable of targeting and lysing a target cell which bears a class I histocompatibility complex on its cell surface and which is infected with an intracellular pathogen. The term xe2x80x9ccytotoxic T cell responsexe2x80x9d is understood to mean any cytotoxic activity that is mediated by cytotoxic T cells.
As used herein, the term xe2x80x9cintracellular pathogenxe2x80x9d is understood to mean any viable organism, including, but not limited to, viruses, bacteria, fungi, protozoa and intracellular parasites, capable of existing within a mammalian cell and causing a disease in the mammal.
In a preferred aspect of the invention, the stress protein-peptide vaccines have particular utility in treating human diseases caused by intracellular pathogens. It is contemplated that the vaccines developed using the principles described herein will be useful in treating diseases of other mammals, for example, farm animals including: cattle; horses; goats; sheep; and pigs, and household pets including: cats; and dogs.
Vaccines may be prepared that stimulate cytotoxic T cell responses against cells infected with viruses including, but not limited to, hepatitis type A, hepatitis type B, hepatitis type C, influenza, varicella, adenovirus, herpes simplex type I (HSV-I), herpes simplex type II (HSV-II), rinderpest, rhinovirus, echovirus, rotavirus, respiratory synctial virus, papilloma virus, papova virus, cytomegalovirus, echinovirus, arbovirus, huntavirus, coxsachie virus, mumps virus, measles virus, rubella virus, polio virus, human immunodeficiency virus type I (HIV-I), and human immunodeficiency virus type II (HIV-II). Vaccines also may be prepared that stimulate cytotoxic T cell responses against cells infected with intracellular bacteria, including, but not limited to, Mycobacteria, Rickettsia, Mycoplasma, Neisseria and Legionella. Vaccines also may be prepared that stimulate cytotoxic T cell responses against cells infected with intracellular protozoa, including, but not limited to, Leishmania, Kokzidioa, and Trypanosoma. Vaccines may be prepared that stimulate cytotoxic T cell responses against cells infected with intracellular parasites including, but not limited to, Chlamydia and Rickettsia.
In another preferred embodiment of the invention, the stress protein peptide vaccine may also contain a therapeutically effective amount of a cytokine. As used herein, the term xe2x80x9ccytokinexe2x80x9d is meant to mean any secreted polypeptide that influences the function of other cells mediating an immune response. Currently, preferred cytokines include: interleukin-1xcex1(IL-1xcex1), interleukin-1xcex2(IL-1xcex2), interleukin-2 (IL-2), interleukin-3 (IL-3), interleukin4 (IL-4), interleukin-5 (IL-5), interleukin-6 (IL-6), interleukin-7 (IL-7), interleukin-8 (IL-8), interleukin-9 (IL-9), interleukin-10 (IL-10), interleukin-11 (IL-11), interleukin-12 (IL-12), interferon a (IFNxcex1), interferon xcex2(IFNxcex2), interferon xcex3, (IFNxcex3), tumor necrosis factor xcex1(TNFxcex1)), tumor necrosis factor xcex2(TNFxcex2), granulocyte colony stimulating factor (G-CSF), granulocyte/macrophage colony stimulating factor (GM-CSF), and transforming growth factor xcex2(TGF-xcex2). It is contemplated that other but as yet undiscovered cytokines may be effective in the invention. In addition, conventional antibiotics may be co-administered with the stress protein-peptide complex. The choice of a suitable antibiotic or a combination thereof, however, will be dependent upon the disease in question.
It has been discovered that the vaccine stimulates the cytotoxic T cell response via the major histocompatibility complex (MHC) class I cascade. Thus, it is contemplated that the cytotoxic T cell response may be enhanced further by co-administering the vaccine with a therapeutically effective amount of one or more of cytokines that potentiate or modulate cytotoxic T cell responses.
Another preferred embodiment, the invention provides a method for stimulating in a mammal a cellular immune response, specifically a cytotoxic T cell response, against cells infected with a preselected intracellular pathogen. The method involves administering to the mammal a vaccine made in accordance with the principles disclosed herein in an amount sufficient to elicit in the mammal a cytotoxic T cell response against the preselected intracellular pathogen.
The vaccine may be administered prophylactically to a mammal in order to stimulate in the mammal a cytotoxic T cell response that prevents subsequent infection of the mammal by the intracellular pathogen. Alternatively, the vaccine may be administered therapeutically to a mammal having a disease caused by an intracellular pathogen. It is contemplated that the vaccine may stimulate a cytotoxic T cell response against cells presently infected with the intracellular pathogen.
The dosage and means of administration of the family of stress protein-peptide vaccines necessarily will depend upon the nature of the complex, the intracellular pathogen and the nature of the disease in question. The complex should be administered in an amount sufficient to initiate a cytotoxic T cell response against the intracellular pathogen. In general, the amount of stress protein-peptide complex administered may range from about 0.1 to about 1000 micrograms of complex/kg body weight of the mammal/immunization, and preferably in the range of about 0.5 to 100 micrograms of complex/kg body weight of the mammal/immunization. The recipient preferably should be vaccinated four times at weekly intervals. If necessary, the responses may be boosted at a later date by subsequent administration of the vaccine. It is contemplated, however, that the optimal dosage and vaccination schedule may be determined empirically for each stress protein-peptide vaccine complex by an artisan using conventional techniques well known in the art.
In another aspect, the invention provides a variety of methodologies for preparing commercially available amounts of the stress-protein peptide vaccines which when administered to a mammal induce in the mammal a cytotoxic T cell response against cells infected with a preselected antigen. In one approach, the stress protein-peptide complex may be harvested using conventional protein purification methodologies from a sample of tissue, an isolated cell or immortalized cell line infected with the preselected intracellular pathogen, or an isolated cell or immortalized cell line transfected with, and expressing a gene encoding a preselected antigenic determinant. The purified complex subsequently may be stored or combined with a pharmaceutically acceptable carrier for administration as a vaccine.
Alternatively, the stress protein-peptide complex may be prepared by reconstituting a potentially antigenic peptide and a stress protein in vitro. For example, the antigenic peptide may be eluted from either a purified stress protein-peptide complex or a MHC-peptide complex using methodologies well known in the art. Specifically, the peptides may be eluted from the stress protein-peptide complex by incubating the complex in the presence of ATP or low pH. Alternatively, the peptides may be eluted from the MHC-peptide complex by incubating the complex in the presence of trifluoroacetic acid (TFA). The resulting peptides may be purified by reverse phase HPLC and their amino acid sequences determined by standard protein sequencing methodologies. Peptides of defined sequence then may be synthesized using conventional peptide synthesis methodologies. Stress proteins may be purified directly from cells naturally expressing the stress proteins. Alternatively, recombinant stress proteins, including non native forms, truncated analogs, muteins, fusion proteins as well as other constructs capable of mimicking the peptide binding and immunogenic properties of stress proteins may be expressed using conventional recombinant DNA methodologies. For example, a recombinant stress protein may be expressed from recombinant DNA in either a eukaryotic or prokaryotic expression system and purified from the expression system. The two purified components then may be combined in vitro to generate a synthetic and completely defined stress protein-peptide complex. The immunogenicity and specificity of the recombinant complexes subsequently may be assayed in vitro and in vivo to identify useful candidate complexes that stimulate cytotoxic T cell responses against a preselected intracellular pathogen. Once identified, the synthetic complexes may be prepared on any scale, stored as is, or combined with pharmaceutically acceptable carriers for administration to mammals.